Molecular Identification and Antifungal Susceptibility Pattern of Non-albicans Candida Species Isolated from Vulvovaginal Candidiasis

Background: Vulvovaginal candidiasis (VVC) is an important health problem caused by Candida spp. The aim of this study was molecular identification, phylogenetic analysis, and evaluation of antifungal susceptibility of non-albicans Candida isolates from VVC. Methods: Vaginal secretion samples were collected from 550 vaginitis patients at Sayyad Shirazi Medical and Educational Center of Gorgan (Golestan Province, Iran) from May to October 2015. Samples were analyzed using conventional mycological and molecular approaches. Clinical isolates were analyzed with specific PCR using CGL primers, and the internal transcribed spacer region and the D1-D2 domain of the large-subunit rRNA gene were amplified and sequenced. Susceptibility to amphotericin B, fluconazole, itraconazole, and clotrimazole was determined by the guidelines of the Clinical and Laboratory Standard Institute. Results: In total, 35 non-albicans Candida isolates were identified from VVC patients. The isolates included 27 strains of Candida glabrata (77.1%), 5 Candida krusei (Pichia kudriavzevii; 14.3%), 2 Candida kefyr (Kluyveromyces marxianus; 5.7%), and 1 Candida lusitaniae (Clavispora lusitaniae; 2.9%). The fungicides itraconazole and amphotericin B were effective against all species. One isolate of C. glabrata showed resistance to fluconazole and clotrimazole, and 26 isolates of C. glabrata indicated dose-dependent susceptibility to fluconazole. C. lusitaniae was susceptible in a dose-dependent manner to fluconazole and resistant to clotrimazole. Conclusions: Non-albicans Candida spp. are common agents of vulvovaginitis, and C. glabrata is the most common species in the tested patients.


INTRODUCTION
he incidence of vulvovaginal candidiasis (VVC) caused by non-albicans Candida spp. has increased considerably [1,2] . Based on evidence, about two-thirds of women worldwide have experienced at least one episode of VVC during their lifetime [3] and some with recurrent episodes [1] . Recurrent episodes are more often caused by nonalbicans Candida spp. against which azole antifungal T agents show low effectiveness [4] .
Candida albicans is the major cause of vulvovaginitis, and Candida glabrata and Candida tropicalis appear to be the most common non-albicans Candida species involved in this disease [2,[4][5][6] . Identification of many species of Candida by traditional methods is a challenge and is sometimes imprecise, particularly for uncommon microorganisms. Sequence analysis of the internal transcribed spacer (ITS) region of the rRNA gene and the D1-D2 domain of the large-subunit rRNA gene as well as PCR-RFLP on the ITS region have been used extensively for identification of fungal pathogens [7][8][9][10][11] .
Minimal inhibitory concentrations (MIC) of azoles against some strains of non-albicans Candida species are high, due to intrinsic resistance [12] . The antifungal susceptibility of Candida spp. causing VVC varies [12,13] , and reliable identification and assessment of drug sensitivity of Candida vaginal isolates are of value in determining proper treatment. The aim of this study was molecular identification, phylogenetic analysis, and evaluation of antifungal susceptibility of non-albicans Candida isolates causing VVC.

Patients and specimens collection
This study was conducted on 550 non-pregnant vaginitis patients referred to Sayyad Shirazi Medical and Educational Center of Gorgan (Golestan, Iran) from May to October 2015. Non-pregnant patients were entered to the study by a simple random sampling method. A questionnaire was completed for each patient about their age, the medical condition (recent antibiotic or antifungal therapy, urinary tract infections, immunodeficiency, diabetes, experiencing at least one episode or recurrent episodes of VVC), and other conditions. Specimens were obtained from vaginal mucosal discharge with a sterile cotton swab. The research protocol was approved by the Ethics Committee of Iran University of Medical Sciences (Tehran, Iran), under Ethics Committee number 93-04-198-25289.

Yeast identification
Microscopic examination was carried out to distinguish yeast forms or pseudohyphae. All samples were cultured on CHROMagar Candida (CHROMagar, France) for identification of mixed infections of Candida spp. [14] . The isolates were identified by carbohydrate assimilation method using API 20C AUX system (Biomérieux, France) [15] .

DNA extraction
A single colony of each clinical isolate from CHROMagar Candida was subcultured on yeast extract peptone dextrose agar and incubated at 37 °C for 24-48 h. Genomic DNA was extracted from yeast cultures using the Qiagen DNA tissue kit (Germany). The extracted DNA was stored at -20 °C for further use.

Specific PCR
All clinical isolates with mauve, pink, or white colonies on CHROMagar Candida as well as C. glabrata CBS 138, as the reference strain, were analyzed by C. glabrata-specific PCR with CGL1-(5´-TTA TCA CAC GAC TCG ACA CT-3´) and CGL2-(5´-CCC ACA TAC TGA TAT GGC CTA CAA-3´) [7] primers. The PCR thermal cycles were as follows: an initial denaturation at 96 °C for 5 min followed by 40 cycles of 30 s at 94 °C, 30 s at annealing temperature of 58 °C and 30 s at 72 °C. A final extension of 15 min at 72 °C was included at the end of PCR cycles.

Phylogenetic analysis
The sequencing results of the D1-D2 and the ITS domains were analyzed and compared with the reference strains by neighbor-joining method using MEGA 7 (TreeView software).

Antifungal drug susceptibility testing
Tests of susceptibility to amphotericin B, fluconazole, clotrimazole, and itraconazole (Sigma, Germany) were conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (document M27-S3 and S4) [17,18] . C. glabrata CBS 138 was used as the reference strain, and all tests were duplicated.

RESULTS Patients
A total of 550 vaginal specimens of non-pregnant vaginitis patients were studied. Individuals with conditions such as infection by Trichomonas vaginalis, Mycoplasma urealyticum, or Chlamydia, as well as bacterial vaginosis or vulval skin disease were excluded from the study. In addition, 122 (22.2%) nonpregnant vaginitis patients showed VVC, and C. albicans isolates were identified in 87 (71.3%) VVC patients (data not shown). Non-albicans Candida isolates were found in 35 (28.7%) VVC patients aged 19-39 years from Gorgan (Table 1). All patients were negative for diabetes, immunodeficiencies, or any chronic disease and were not taken any antifungal treatment.

Amplification with specific primers
The clinical isolates with mauve, pink, or white colonies on CHROMagar Candida and C. glabrata CBS 138 were analyzed with CGL1/2 specific primers, and the presence of the 423-bp fragment amplified with these primers confirmed those isolates identical to C. glabrata (Fig. 1).

PCR amplification and sequencing of ITS region and D1-D2 domain
PCR amplification of all clinical isolates with ITS1 and ITS4 primers yielded the fragments of 350-880 bp. The ITS fragments of C. glabrata were ~500 to ~879 bp (Fig. 2) [8,10] , while C. krusei, C. kefyr, and C. lusitaniae yielded the fragments of ~500, ~720, and ~370 bp, respectively (Fig. 2) [8] . The ITS fragments of three clinical C. glabrata isolates showing ~500 and ~600 bp were compared to the reference data in the GenBank database using the BLAST. Three C. glabrata isolates showed partial sequences of ITS region (~500 and ~600 bp), while the complete sequences of ITS region of C. glabrata was ~879 bp, and the partial sequences and complete sequences were submitted to the GenBank ( Table 2). The D1-D2 region of the large-subunit rRNA gene amplified with NL1 and NL4 primers yielded the fragments of ~600 bp (Fig. 3). The ITS and D1-D2 region sequences of non-albicans Candida clinical isolates were compared to the reference data in the GenBank database using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). All clinical isolates were correctly determined to species level. The sequences were submitted to the GenBank under accession numbers KU845721, KU904424-26, KU992386-95, KX008737-53, and KX016018-21 ( Table 2).

Antifungal drug susceptibility
Results of susceptibility testing of the 35 nonalbicans Candida isolates showed one isolate of C. glabrata to be resistant to fluconazole (MIC ≥ 64 µg/ml) and clotrimazole (MIC ≥ 4 µg/ml), and 26 isolates of C. glabrata were susceptible to fluconazole (MIC ≤ 32 µg/ml) in a dose-dependent manner [18] . Single clinical isolate of C. lusitaniae showed dosedependent susceptibility to fluconazole (MIC = 16-32 µg/ml) and resistant to clotrimazole (MIC = 2 µg/ml) [17] . The MICs for one isolate of C. krusei were as follows: fluconazole ≤ 32 µg/ml and clotrimazole = 2 µg/ml. Because the clinical isolates of C. krusei showed intrinsic resistant to fluconazole, and their MICs should not be interpreted using this scale; therefore, breakpoint was not provided by CLSI document M27-S4 [18] . Itraconazole and amphotericin B were active against all of the isolates (Table 3).

DISCUSSION
This study revealed that non-albicans Candida spp., as important agents, are commonly associated with vulvovaginitis; C. glabrata is the second in rate of occurrence after C. albicans. Other investigations have found that C. glabrata and C. albicans to be the most common species isolated from VVC patients [13,19,20] . The overall proportion of non-albicans infection in vaginitis has been reported to be high [2] . C. glabrata and C. tropicalis have also been found in the normal vaginal flora of women in China [21] . Infections caused by less common yeasts have been increasingly observed [22] , and identification of a variety of medically important yeast species by traditional approaches may be challenging. Molecular methods can improve discrimination of uncommon clinical isolates and closely related yeast species such as those in Candida complexes. Molecular diagnostics are also useful in carrying out large epidemiological studies of pathogenic yeasts. In this study, conventional methods and specific PCR with CGL primers were used for identification of C. glabrata. Sequencing the ITS and D1-D2 regions has proven to be a feasible method for the reliable identification of clinically important yeasts,  (Table 1) and reference strains (KU729202 and KU72920).  5. Molecular phylogenetic analysis using Neighbor-Joining method with sequences of ITS region. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Bootstrap values greater than 70% from 1000 replicates are indicated at the nodes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA7.
principally the C. glabrata complex (C. glabrata, C. bracarensis, and C. nivariensis) [22,23] and the C. parapsilosis complex (C. parapsilosis, C. orthopsilosis, and C. metapsilosis) [24] . Richter et al. [13] reported that 173 of 593 yeast isolates from vaginitis patients were non-albicans Candida spp., and that C. albicans was the most frequent cause of vaginal candidiasis, followed by C. glabrata, C. parapsilosis, C. krusei, Saccharomyces cerevisiae, C. tropicalis, and C. lusitaniae [13] . Vijaya et al. [25] showed that C. tropicalis is the major non-albicans species of Candida associated with vaginal candidiasis. Other studies in Iran introduced C. glabrata as the most important nonalbicans species in vaginal candidiasis patients [19,20] . Shi et al. [26] demonstrated that C. albicans is the main cause of vaginal candidiasis, followed by C. glabrata,  C. tropicalis and C. parapsilosis in China. In the current study, C. glabrata was the most common species of non-albicans Candida. Candida spp., especially C. glabrata and C. albicans, represent a primary source of infection leading to bloodstream infections and to morbidity and mortality in severely affected and immune-compromised individuals [12,[27][28][29] . Species C. krusei and C. glabrata have been indicated.
To be resistant or to have low susceptibility to azole drugs [4,12] . C. lusitaniae has also been shown to have resistance to amphotericin B, caspofungin, and azoles [30] . A study in Japan revealed that one of the 19 C. glabrata clinical isolates of VVC patients showed resistance to fluconazole, and this isolate demonstrated cross-resistance to other antimycotic drugs tested [31] .
Another study displayed that non-albicans isolates, particularly C. glabrata strains, were susceptible in a dose-dependent manner and were resistant to fluconazole [13] . Kalkanci et al. [32] suggested that C. glabrata was the most vaginal isolate of non-albicans Candida, and 3 of 81 (3.7%) C. glabrata isolates were resistant to ketoconazole, and only one C. glabrata was fluconazole resistant. Also, five C. glabrata isolates showed susceptibility to fluconazole in a dosedependent manner. A previous study indicated that nystatin was an appropriate option instead of imidazoles [33] . An investigation from Iran found that clinical isolates of Candida spp. were susceptible to clotrimazole, miconazole, and nystatin [19] . Razzaghi-Abyaneh et al. [34] indicated that itraconazole was the most effective antimycotic drug for C. krusei, C. glabrata, and C. guilliermondii isolates of superficial candidiasis in Iran. In the current study, 26 isolates of C. glabrata were susceptible dose-dependent to fluconazole, and one isolate was resistant to fluconazole and clotrimazole. In addition, one isolate of C. lusitaniae was susceptible to fluconazole in a dose-dependent manner and resistance to clotrimazole [35,36] .
Sequencing of the ITS region and D1-D2 domain appears to be the most effective method for identification of Candida spp. The phylogenetic trees based on sequences of D1-D2 and ITS domains showed similarity of Candida spp. to closely related reference species. Results suggested that amphotericin B and itraconazole retain good clinical effectiveness. Accurate identification and assessment of susceptibility of Candida spp. isolates are critical to treatment management, since some strains showed varying degrees of resistance to antifungal drugs.